Subtilisin is a protease having a wide range of applications including use in laundry detergents, silver recovery, manufacturing of fish meal and other products and processes. The crystalline form of subtilisin is preferred for a number of reasons. Crystalline subtilisin is of higher purity than other forms of subtilisin produced by other routes, such as precipitation of amorphous subtilisin. Subtilisin crystals afford a high degree of stability as well as flexibility in choices of media and formulation for various desired end uses. Subtilisin, whether in crystalline or in precipitated amorphous form, can be dried and used in various granular or powdered products. However, it is more often dissolved and formulated in the form of various liquid products, e.g., in aqueous, glycerin, sorbitol, propylene glycol, etc., solutions, for use in liquid products or for use in manufacturing of liquid or solid products. Because of its higher purity, crystalline subtilisin and liquid products made from the crystalline form have a number of high grade uses, such as in medical applications and in food grade products.
The subtilisin crystals themselves are rhomboidal plates usually ranging in size from about 5 to about 50 microns. The crystals have a solubility of about 4 grams per liter(g/l) in water, about 8 g/l in brine, and over about 500 g/l in propylene glycol.
Subtilisin crystals have been obtained from fermentation of Bacillus subtilis in much the same way as crystals of other enzymes are obtained from given fermentations. A fermentation mixture is first subjected to a separation step which removes bacterial cells and suspended solids to form a resulting solution or broth free of solids. The resulting solution is often concentrated to provide a certain minimum concentration level of the desired enzyme. Crystallization is then carried out, usually with the aid of seed crystals, and the high purity crystalline product separated from the solution.
As discussed in "Preparation And Crystallization Of The Enzymes," from Northrup, et al., Crystalline Enzymes, (1948) Columbia Vine Press, New York, New York, at pp. 253-254, concentrated protein solutions, i.e., 1-10 percent, are essential to protein crystal production, because proteins in dilute protein solutions can be separated only with difficulty, if at all, whereas the same proteins in concentrated solution are typically much easier to separate. It is also necessary to crystallize the protein without forming precipitates which result in the less pure amorphous form. The crystallization is induced by adjusting the relative concentrations of protein, salt, solvent and/or organic solvents in the solution. It is important that the solution not be saturated or too highly supersaturated in salt or solvent since supersaturation favors the formation of the less desirable amorphous form of the protein by precipitation. Therefore, an optimal condition for obtaining a crystalline enzyme product has been a concentrated salt/enzyme solution at the level of very slight subsaturation.
Such optimal conditions of slight supersaturation are frequently difficult to achieve and particularly difficult to maintain during processing using conventional crystallization techniques. More specifically, the usual procedure for crystallization of enzymes involves adding a precipitating agent, such as ammonium sulfate or sodium sulfate, until a precipitate just appears. However, a precipitate frequently does not appear until the solution is too highly supersaturated and thus, the process has always involved a risk that the amorphous form of the enzyme would be produced by precipitation.
Even in instances where the optimal slight supersaturation is achieved, such ideal conditions rapidly become non-ideal. More specifically, by removing protein from solution as the crystals form, the remaining solution loses its near saturation concentration and it becomes increasingly difficult for more crystals to form. This leads to considerable losses of protein product since much of the protein may be left behind in the supernatant. To overcome this problem, it is necessary to continually adjust the solution conditions by adding additional amounts of salts such that the solution can be kept in a slightly supersaturated state until nearly all the protein has crystallized therefrom.
As is apparent from the above discussion, currently known techniques for the preparation of crystalline subtilisin are often unreliable and difficult to control. Not only must the practitioner be able to determine the point at which a subtilisin solution is slightly supersaturated but once crystallization commences, such practitioner must also continually adjust the solution system to maintain the desired concentration to avoid loss of the valuable subtilisin product and to avoid any too highly supersaturated conditions and the consequent formation of the amorphous form of the subtilisin.
In view of the foregoing limitations and shortcomings of known enzymatic crystallization techniques as well as other disadvantages not mentioned above, it is apparent that there is a need for a process for purifying subtilisin by crystallization which does not depend on the maintenance of a slightly supersaturated concentration of the enzyme solution for crystallization to occur and which enables efficient and reliable preparation of crystalline subtilisin. It is an object of this invention to fulfill that need by providing a process for purifying subtilisin enzyme by crystallization using salts which induce crystallization of the subtilisin enzyme when said enzyme solution is supersaturated thereby avoiding formation of the amorphous precipitates which can form when the subtilisin enzyme solution is highly supersaturated.
It is a further object of the present invention to provide a process for purifying subtilisin enzyme by crystallization which does not require ammonium sulfate as the precipitating agent, and thereby avoids the environmental and waste disposal problems attendant with ammonium sulfate use.
Another object of the present invention is to provide a process for the purification of subtilisin by crystallization which has low raw material and operating costs.
Another object of the present invention is to provide a process for the purification of subtilisin by crystallization which has the capability of high yields and high throughputs.
Another object of the present invention is to provide a process for the purification of subtilisin by crystallization which produces a subtilisin product which is of high purity and which is virtually free of microorganisms.
With the foregoing and other objects, advantages, and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following description of the invention, the appended claims and the drawings.